Parameciumswimming and ciliary beating patterns: a study on four RNA interference mutations

Abstract

Paramecium cells swim and feed by beating their thousands of cilia in coordinated patterns. The organization of these patterns and its relationship with cell motility has been the subject of a large body of work, particularly as a model for ciliary beating in human organs where similar organization is seen. However the rapid motion of the cells makes quantitative measurements very challenging. Here we provide detailed measurements of the swimming of Paramecium cells from high-speed video at high magnification, as they move in microfluidic channels. An image analysis protocol allows us to decouple the cell movement from the motion of the cilia, thus allowing us to measure the ciliary beat frequency (CBF) and the spatio-temporal organization into metachronal waves along the cell periphery. Two distinct values of the CBF appear at different regions of the cell: most of the cilia beat in the range of 15 to 45 Hz, while the cilia in the peristomal region beat at almost double the frequency. The body and peristomal CBF display a nearly linear relation with the swimming velocity. Moreover the measurements do not display a measurable correlation between the swimming velocity and the metachronal wave velocity on the cell periphery. These measurements are repeated for four RNAi silenced mutants, where proteins specific to the cilia or to their connection to the cell base are depleted. We find that the mutants whose ciliary structure is affected display similar swimming to the control cells albeit with a reduced efficiency, while the mutations that affect the cilia's anchoring to the cell lead to strongly reduced ability to swim. This reduction in motility can be related to a loss of coordination between the ciliary beating in different parts of the cell.

TetrahymenaRIB72A and RIB72B are Microtubule Inner Proteins in the ciliary doublet microtubules

“…The metachronal waves were visible near the anterior end of the wild type chronograph, as dark diagonal lines ( Fig. 4B) (Funfak et al, 2015). The metachronal waves were visible but less frequent in the single KO strains but completely absent in the double knockout cells ( Fig.…”

“…To analyze cilia beating in live cells, we collected high-speed videos of forward-swimming cells that were partially restrained inside microfluidic channels (Videos S1-S5). Using an image processing workflow that was originally developed for Paramecium (Funfak et al, 2015), we extracted video segments that contain the zone of beating cilia around the entire cell circumference as outlined in Fig. 4A.…”

“…Observations of cilia in actively-swimming Tetrahymena cells were performed and data were quantified as described previously for Paramecium tetraurelia (Funfak et al, 2015), with minor modifications. A highly concentrated cell suspension (in 2% SPP medium) was injected into the inlet of a microfluidic channel (33 μm in height and 50 μm in width) using a controllable pressure source (Fluigent).…”

“…Videos were recorded on a Photron 1024PCI camera at 2000 frames per second. Video image processing and quantification was done exactly as described (Funfak et al, 2015). Matlab (The MathWorks Inc., Natick, MA) along with the CR Toolbox (Adelin Barbacci, 2014) were used to mark the cell body and the zone of beating cilia in the first frame of the movie.…”

Influence of cellular shape on sliding behavior of ciliates

“…One of the characteristic features of ciliates is the presence of cilia on the entire cell surface, whose beating motions induce thrust force to swim in water. Ciliates exhibit remarkable behaviors using cilia, such as helical swimming in bulk water [3–5] and accumulation on air/fluid and solid/fluid interfaces [6,7]. Since nutrition is deposited on the interfaces, it is advantageous that ciliates prefer the interfaces.…”